The synthesis and degradation of basement membrane components will be compared in normal and diabetic tissues. The synthesis of four distinct proteins will be studied in tissue cultures of the EHS sarcoma which synthesizes a basement membrane matrix. In the case of each protein, tumor tissue from both normal and diabetic mice will be incubated in vitro with or without insulin and with varying glucose concentrations. Type IV collagen will be labeled with amino acids and purified. Changes in total collagen synthesis will be studied and also possible changes in amounts of 3-hydroxyproline, 4-hydroxyproline, hydroxylysine and hydroxylysineglycosides in the collagen. The chains will be separated and amino acids studied. Synthesis of fibronectin and laminin will be studied. The proteins will be labeled and purified and quantitated following immunoprecipitation. A basement membrane proteoglycan will be labeled and possible changes in sulfation and glucosamine amounts will be assessed. The activity of a specific type IV collagenase will be studied to see if accumulation of collagen in diabetic basement membrane is caused by a decreased degradation. Studies on the cell-free translation of basement membrane components will be extended to measure the levels of mRNA for the components in normal and diabetic tissue. The mRNA will be extracted and purified by oligo(dT) chromatography and ultracentrifugation on sucrose gradients followed by translation using reticulocyte lysate. The translation products will be identified and quantitated using immunoprecipitation with specific purified antibodies. Attempts will be made to prepare cloned DNA to this mRNA in collaboration with Dr. Peter Muller, Max Planck, Munich. The goal of this study is to find out how basement membranes are altered in diabetes mellitus and if the changes can be corrected with insulin and glucose administration. Furthermore this research could result in the development of sensitive methods for detecting and assessing changes leading to microangiopathy.